Advertisement

Basic Research in Cardiology

, Volume 85, Issue 4, pp 411–427 | Cite as

Quantification of the total Na,K-ATPase concentration in atria and ventricles from mammalian species by measuring3H-ouabain binding to intact myocardial samples. Stability to short term ischemia reperfusion

  • T. A. Schmidt
  • J. H. Svendsen
  • S. Haunsø
  • K. Kjeldsen
Original Contributions

Summary

Na,K-ATPase concentration was measured by vanadata facilitated3H-ouabain binding to intact samples taken from various parts of porcine and canine myocardium. In porcine and canine heart3H-ouabain binding site concentration in ventricles was 1.4–2.5 times larger than in atria. Evaluation of3H-ouabain binding kinetics revealed no major difference between atria and ventricles: Equilibrium was obtained after the same incubation time in right atrium (RA) as in left ventricle (LV), both in porcine and canine heart. Unspecific uptake and retention of3H-ouabain was for porcine heart RA and LV 1.5 and 1.4, respectively, and for canine heart RA and LV, both 1.2% filling (i.c., volume (ml) of incubation medium3H-radioactivity taken up per mass unit (g wet wt.) of tissue multiplied by 100). The apparent dissociation constant (K d ) was 1.4×10−8 and 1.9×10−8 in porcine RA and LV and 2.6×10−8 and 6.1×10−8 mol/l in canine RA and LV. Loss of specifically bound3H-ouabain during the washout procedure occurred with a half-life time (T1/2) of 16.7 in RA and LV of porcine heart and 91.2 and 151.6h in RA and LV of canine heart. Duly corrected for these errors of the method-factor 1.16 and 1.13, respectively, for porcine RA and LV, and factor 1.11 and 1.13 for canine RA and LV, total3H-ouabain binding site concentration was found to be 553±74 and 1037±45 pmol/g wet wt. (means±SEM, n=5) in porcine RA and LV, and 569±37 and 1410±40 pmol/g wet wt. (means ±SEM, n=5) in the canine RA and LV. These values were confirmed by measurements of3H-digoxin binding to the porcine heart. The present quantification of myocardial Na, K-ATPase gives values up to 154 times higher than measurements based upon Na,K-ATPase activities in membrane fractions where the recovery of Na,KK-ATPase may be less than 1% due to loss during purification. A higher Na,K-ATPase concentration is found in small animals than in large animals. A relationship between higher concentration of Na, K-ATPase and larger pressure work in ventricles compared to atria is suggested. Myocardial3H-ouabain binding sites were found to be stable for 20 min of ischemia, followed by 1h of reperfusion, supporting the concept that myocyte injury induced by short term ischemia may be reversible and that reperfusion may result in normalization.

Key words

Na,K-ATPase 3H-ouabain binding sites cardiac glycosides myocardium ischemia 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Beller GA, Conroy J, Smith TW (1976) Ischemia-induced alteration in myocardial (Na+-K+)-ATPase and cardiac glycoside binding. J Clin Invest 57:341–350PubMedGoogle Scholar
  2. 2.
    Charlemangne D, Mayoux E, Poyard M, Oliviero P, Geering K (1987) Identification isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart. J Biol Chem 262:8941–8943PubMedGoogle Scholar
  3. 3.
    Chemnitius JM, Sasaki Y, Burger W, Bing RJ (1985) The effect of ischemia and reperfusion on sarcolemmal function in perfused canine hearts. J Mol Cell Cardiol 17:1139–1150PubMedGoogle Scholar
  4. 4.
    Clausen T, Everts ME, Kjeldsen K (1987) Quantification of the maximum capacity for active sodium-potassium transport in rat skeletal muscle. J Physiol Lond 388:163–181PubMedGoogle Scholar
  5. 5.
    Clausen T, Hansen O (1974) Ouabain binding and Na+-K+-transport in rat muscle cells and adipocytes. Biochim Biophys Acta 345:387–404Google Scholar
  6. 6.
    Clausen T, Sellin LC, Thesleff S (1981) Quantitative changes in ouabain binding after denervation and during reinnervation of mouse skeletal muscle. Acta Physiol Scand 111:373–375PubMedGoogle Scholar
  7. 7.
    Godin DV, Bhimji S (1987) Effects of allopurinol on myocardial ischemic injury induced by coronary artery ligation and reperfusion. Biochem Pharmacol 36:2101–2107PubMedGoogle Scholar
  8. 8.
    Hansen O (1979) Facilitation of ouabain binding to (Na++K+)-ATPase by vanadate at in vivo concentrations. Biochim Biophys Acta 568:265–269PubMedGoogle Scholar
  9. 9.
    Hansen O (1984) Interaction of cardiac glycosides with (Na+ +K+-activated ATPase. A biochemical link to digitalis-induced inotropy. Pharmacol Rev 36:143–163PubMedGoogle Scholar
  10. 10.
    Hansen O, Clausen T (1988) Quantitative determination of Na+-K+-ATPase and other sarcolemmal components in muscle cells. Am J Physiol 254:C1-C7PubMedGoogle Scholar
  11. 11.
    Hansen O, Skou JC (1973) A study on the influence of the concentration of Mg++, Pi, K+, Na+, and tris on (Mg++ +Pi)-supported g-strophanthin binding to (Na++K+)-activated ATPase from ox brain. Biochim Biophys Acta 311:51–66PubMedGoogle Scholar
  12. 12.
    Harper JS, Lochner A (1989) Sarcolemmal integrity during ischaemia and reperfusion of the isolated rat heart. Basic Res Cardiol 84:208–226PubMedGoogle Scholar
  13. 13.
    Jones LR, Besch HR (1984) Isolation of canine cardiac sarcolemmal vesicles. Meth Pharmacol 5:1–12Google Scholar
  14. 14.
    Jones LR, Maddock SW, Besch HR (1980) Unmasking effect of alamethicin on the (Na+, K+-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles. J Biol Chem 255:9971–9980PubMedGoogle Scholar
  15. 15.
    Kim M-S, Akera T (1987) O2 free radicals: Cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase. Am J Physiol 252:H252–H257PubMedGoogle Scholar
  16. 16.
    Kim DH, Akera T, Kennedy RH (1983) Ischemia-induced enhancement of digitalis sensitivity in isolated guinea pig heart. J Pharmacol Exp Ther 226:335–342PubMedGoogle Scholar
  17. 17.
    Kjeldsen K (1986) Complete quantification of the total concentration of rat skeletal muscle Na++K+-dependent ATPase by measurements of (3H)ouabain binding. Biochem J 240:725–730PubMedGoogle Scholar
  18. 18.
    Kjeldsen K (1987) Regulation of3H-ouabain binding sites in mammalian skeletal muscle.-Effects of age, K-depletion, thyroid status and hypertension. Dan Med Bull 34:15–46Google Scholar
  19. 19.
    Kjeldsen K (1988) Homogeneity of3H-ouabain binding sites in rat soleus muscle. Biochem J 249:481–485PubMedGoogle Scholar
  20. 20.
    Kjeldsen K, Bjerregaard P, Richter EA, Thomsen PEB, Nørgaard A (1988) Na+, K+-ATPase concentration in rodent and human heart and skeletal muscle. Apparent relationship to muscle performance. Cardiovasc Res 22:95–100PubMedGoogle Scholar
  21. 21.
    Kjeldsen K, Grøn P (1990) Age dependent change in myocardial cardiac glycoside receptor (Na, K-ATPase) concentration in children. J Cardiovase Pharmacol 15:332–337Google Scholar
  22. 22.
    Kjeldsen K, Nørgaard A, Clausen T (1984) The age-dependent changes in the number of3H-ouabain binding sites in mammalian skeletal muscle. Pflügers Arch 402:100–108Google Scholar
  23. 23.
    Kjeldsen K, Nørgaard A, Hansen O, Clausen T (1985) Significance of skeletal muscle digitalis receptors for3H-ouabain distribution in the guinea pig. J Pharmacol Exp Ther 234:720–727PubMedGoogle Scholar
  24. 24.
    Kjeldsen K, Richter EA, Galbo H, Lortie G, Clausen T (1986) Training increases the concentration of3H-ouabain binding sites in rat skeletal muscle. Biochim Biophys Acta 860:708–712PubMedGoogle Scholar
  25. 25.
    Kloner RA, Braunwald E (1980) Observation on experimental ischaemia. Cardiovasc Res 14:371–395PubMedGoogle Scholar
  26. 26.
    Knochel JP, Blachley JD, Johnson JH, Carter NW (1985) Muscle cell electrical hyperpolarization and reduced exercise hyperkalemia in physically conditioned dogs. J Clin Invest 75:740–745PubMedGoogle Scholar
  27. 27.
    Maixent JM, Charlemagne D, Chapelle B, Lelievre LG (1987) Two Na, K-ATPase isoenzymes in canine cardiac myocytes. J Biol Chem 262:6842–6848PubMedGoogle Scholar
  28. 28.
    Matsui H, Schwartz A (1966) Purification and properties of a highly active ouabain-sensitive Na+, K+-dependent adenosinetriphosphatase from cardiac tissue. Biochim Biophys Acta 128:380–390PubMedGoogle Scholar
  29. 29.
    Nørgaard A, Baandrup U, Larsen JS, Kjeldsen K (1987) Heart Na, K-ATPase activity in cardi hamsters as estimated from K-dependent 3-O-MFPase activity in crude homogenates. J Moll Cell Cardiol 19:589–594Google Scholar
  30. 30.
    Nørgaard A, Bagger JP, Bjerregaard P, Baandrup U, Kjeldsen K, Thomsen FEB (1988) Relation of left ventricular function and Na, K-pump concentration in suspected idiopathic dilated cardiomyopathy. Am J Cardiol 61:1312–1315PubMedGoogle Scholar
  31. 31.
    Nørgaard A, Kjeldsen K, Hansen O (1985) K+-dependent 3-O-methylfluorescein phosphatase activity in crude homogenates of rodent heart ventricle: Effect of K+ depletion and changes in thyroid status. Eur J Pharmacol 113:373–382PubMedGoogle Scholar
  32. 32.
    Nørgaard A, Kjeldsen K, Hansen O, Clausen T, Larsen CG, Larsen FG (1986) Quantification of the3H-ouabain binding site concentration in human myocardium: a postmortem study. Cardiovasc Res 20:428–435PubMedGoogle Scholar
  33. 33.
    Panagia V, Singh JN, Anand-Srivastava MB, Pierce GN, Jasmin G, Dhalla NS (1984) Sarcolemmal alterations during the development of genetically determined cardiomyopathy. Cardiovascular Res 18:567–572Google Scholar
  34. 34.
    Pang DC, Weglicki WB (1977) Cardiac sarcolemma of the hamster. Enrichment of the (Na++K+)-ATPase. Biochim Biophys Acta 465:411–414Google Scholar
  35. 35.
    Schwatz A, Wood JM, Allen JC, Bornet EP, Entman ML, Goldstein MA, Sordahl LA, Suzuki M (1973) Biochemical and morphological correlates of cardiac ischemia. Am J Cardiol 32:46–61PubMedGoogle Scholar
  36. 36.
    Sejersted OM, Andersen FR, Ilebekk A (1985) Potassium balance and ouabain binding sites in intact porcine hearts during isoproterenol infusion. Advances in myocardiology 8:83–95Google Scholar
  37. 37.
    Thomlins B, Harding SE, Kirby MS, Poole-Wilson PA, Williams AJ (1986) Contamination of a cardiac sarcolemmal preparation with endothelial plasma membrane. Biochim Biophys Acta 856:137–143PubMedGoogle Scholar

Copyright information

© Dr. Dietrich Steinkopff Verlag 1990

Authors and Affiliations

  • T. A. Schmidt
    • 1
  • J. H. Svendsen
    • 1
  • S. Haunsø
    • 1
  • K. Kjeldsen
    • 1
  1. 1.Department of Medicine B, Division of Cardiology, RigshospitaletUniversity of Copenhagen School of MedicineCopenhagenDenmark

Personalised recommendations